The current standard of care for hemophilia A is treatment with intravenous FVIII protein infusions, either prophylacticly or during bleeding episodes. Prophylactic treatment, however, is problematic due to limited availability, high costs and concerns for blood-borne diseases due to venous access. Gene transfer with viral vectors provides an attractive alternative therapeutic approach with the potential for long-term correction since only 5% of normal FVIII expression levels provide substantial clinical benefits. Success of clinical trials with adeno (Ad)- and oncoretroviral (MLV) FVIII therapy has been compromised to date by lack of transduction of non-proliferating cells (MLV) and high vector particle immunogenicity (Ad). Lentiviral vectors overcome both problems and our cumulative preclinical studies using Feline Immunodeficiency Virus (FIV)-based lentiviral vectors resulted in persistent and therapeutic FVIII expression levels in the hemophilia mouse model. The next logical steps towards the initiation of clinical trials include a detailed investigation of vector particle interactions with the host's immune system as well as the testing of FIV-based FVIII therapy in a relevant large animal model-the hemophilia dog. Our goal is to generate FIV vectors coding for canine B domain-deleted FVIII (cFVIII) optimized for high expression levels. Optimization of cFVIII expression is modeled after successful modifications of the human FVIII and thus better mimics advanced human FVIII therapy while facilitating detection of serum FVIII protein levels and therapeutic benefit in the dog model. Our second goal is aimed at gaining a better understanding of possible immune responses to the FIV vector particle itself. To date, there has been no published investigation of innate or adaptive immune responses to lentiviral vector components in any animal system. A thorough understanding of the level or extent of any immune responses to FIV particles is an important safety and efficacy concern since FVIII gene transfer therapy likely needs to be re-administered after some time. The timeframe of this proposal allows the production of functional FIV-cFVIII vectors and the testing of first innate immune responses to FIV vector particles pseudotyped with three different envelopes. Future studies beyond the scope of the current proposal will be aimed at investigating FIV-based cFVIII gene transfer in the hemophilia dog while monitoring therapeutic expression levels and adaptive anti-FIV immune responses during repeat administrations.